The major aims of this project are to identify the tissue interactions responsible for specifying the ciliary epithelium and the other tissues of the anterior segment of the embryonic eye. In the past grant period we showed that the formation of the ciliary folds correlated with an abrupt increase in DNA synthesis and cell volume in the non-pigmented cells of the ciliary epithelium. These changes in cell dynamics depended on normal intraocular pressure. An increase in cell number and volume is likely to be a major factor in the expansion of the ciliary epithelium and the subsequent formation of ciliary folds. We also tested and disproved several earlier hypotheses concerning the forces responsible for the formation of the ciliary folds. Our studies suggested that contraction of the developing ciliary muscle may contribute to the formation of the folds. This possibility will be tested during the next grant period. We showed that the cells that form the corneal endothelium express alpha- smooth muscle actin before and during their migration over the primary corneal stroma from the edge of the optic cup. The lens probably is responsible for inducing the expression of this antigen, because lenses transplanted to the posterior of the eye cause adjacent mesenchyme cells to migrate and express alpha-smooth actin. The cells at the margin of the anterior chamber, the prospective trabecular meshwork, also expressed alpha-smooth muscle actin. Experiments were performed to test whether the lens induces cells near the edge of the optic cup to form the ciliary epithelium. These studies were complicated by difficulties with the markers used to identify the developing ciliary epithelium. In the next grant period we will perform several tests to define the interactions that lead to the formation of the ciliary epithelium and the ciliary muscle. The lens will be removed or transplanted to ectopic sites to determine whether the expression of acetylcholinesterase activity, type IX collagen mRNA and specific homeobox sequences is altered in the presumptive ciliary epithelium. As part of this project, we cloned a member of the distal-less family of homeobox-containing genes from the embryonic eye. We will follow the expression of this and other hox genes during eye formation. The role of the lens in specifying the time and location of expression of each hox gene will also be determined. Finally, we will identify genes that are expressed exclusively in the non-pigmented layer of the ciliary epithelium. In future years, we will use the regulatory regions of these genes to target chimeric gene sequences specifically to this tissue. This will allow us to test the role of specific transporters and regulatory molecules in the function of the ciliary epithelium, by targeting "dominant negative" constructs to the ciliary epithelium of transgenic animals.